title: Mutation of the salt bridge-forming residues in the ETV6-SAM domain interface blocks ETV6-NTRK3-induced cellular transformation.
abstract: The ETV6-NTRK3 (EN) chimeric oncogene is expressed in diverse tumor types. EN is generated by a t(12;15) translocation, which fuses the N-terminal SAM (sterile α-motif) domain of the ETV6 (or TEL) transcription factor to the C-terminal PTK (protein-tyrosine kinase) domain of the neurotrophin-3 receptor NTRK3. SAM domain-mediated polymerization of EN leads to constitutive activation of the PTK domain and constitutive signaling of the Ras-MAPK and PI3K-Akt pathways, which are essential for EN oncogenesis. Here we show through complementary biophysical and cellular biological techniques that mutation of Lys-99, which participates in a salt bridge at the SAM polymer interface, reduces self-association of the isolated SAM domain as well as high molecular mass complex formation of EN and abrogates the transformation activity of EN. We also show that mutation of Asp-101, the intermolecular salt bridge partner of Lys-99, similarly blocks transformation of NIH3T3 cells by EN, reduces EN tyrosine phosphorylation, inhibits Akt and Mek1/2 signaling downstream of EN, and abolishes tumor formation in nude mice. In contrast, mutations of Glu-100 and Arg-103, residues in the vicinity of the interdomain Lys-99-Asp-101 salt bridge, have little or no effect on these oncogenic characteristics of EN. Our results underscore the importance of specific electrostatic interactions for SAM polymerization and EN transformation.
authors.affiliation: From the Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia V6T 2B5, Canada.
authors.forename: Naniye
authors.initials: N
authors.lastname: Cetinbas
authors.forename: Helen
authors.initials: H
authors.lastname: Huang-Hobbs
authors.forename: Cristina
authors.initials: C
authors.lastname: Tognon
authors.forename: Gabriel
authors.initials: G
authors.lastname: Leprivier
authors.forename: Jianghong
authors.initials: J
authors.lastname: An
authors.forename: Steven
authors.initials: S
authors.lastname: McKinney
authors.forename: Mary
authors.initials: M
authors.lastname: Bowden
authors.forename: Connie
authors.initials: C
authors.lastname: Chow
authors.forename: Martin
authors.initials: M
authors.lastname: Gleave
authors.forename: Lawrence P
authors.initials: LP
authors.lastname: McIntosh
authors.forename: Poul H
authors.initials: PH
authors.lastname: Sorensen
mesh_terms: D000818:Animals; D002151:Calorimetry; D002471:Cell Transformation, Neoplastic; D006801:Humans; D008239:Lysine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008819:Mice, Nude; D009154:Mutation; D041681:NIH 3T3 Cells; D011108:Polymers / Q000737:chemistry; D017434:Protein Structure, Tertiary; D050783:Proto-Oncogene Proteins c-ets / Q000737:chemistry* / Q000235:genetics; D020812:Receptor, trkC / Q000737:chemistry* / Q000235:genetics; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics; D012492:Salts / Q000737:chemistry; D015398:Signal Transduction; D055672:Static Electricity; D014443:Tyrosine / Q000737:chemistry; D000097764:ETS Translocation Variant 6 Protein
chemical_list: D008239:Lysine; D011108:Polymers; D050783:Proto-Oncogene Proteins c-ets; D020812:Receptor, trkC; D012097:Repressor Proteins; D012492:Salts; D014443:Tyrosine; D000097764:ETS Translocation Variant 6 Protein
keywords: Cancer Biology; Cell Signaling; ETV6-NTRK3; Ets Family Transcription Factor; Fusion Oncogene; Fusion Protein; Oncogene; SAM Domain